Experimental Lung Research

ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20

Hydrogen Peroxide Release from Alveolar Macrophages and Alveolar Type II Cells During Adaptation to Hyperoxia in Vivo V. L. Kinnula, L. Y. Chang, Y. S. Ho & J. D. Crapo To cite this article: V. L. Kinnula, L. Y. Chang, Y. S. Ho & J. D. Crapo (1992) Hydrogen Peroxide Release from Alveolar Macrophages and Alveolar Type II Cells During Adaptation to Hyperoxia in Vivo, Experimental Lung Research, 18:5, 655-673, DOI: 10.3109/01902149209031700 To link to this article: http://dx.doi.org/10.3109/01902149209031700

Published online: 02 Jul 2009.

Submit your article to this journal

Article views: 1

View related articles

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ielu20 Download by: [Imperial College London Library]

Date: 01 April 2016, At: 19:35

Hydrogen Peroxide Release from Alveolar Macrophages and Alveolar Type I1 Cells During Adaptation to Hyperoxia in Vivo

Experimental Lung Research 1992.18:655-673.

V. L. Kinnula, L. Y. Chang, Y. S. Ho, and J. D. Crapo

ABSTRACT: The effect of hyperoxia (1-14 days, 85% 0 3 on rat alveolar macrophage and alveolar type 11 cell oxidant and antioxidant characteristics was investigated. Unstimulated control macrophages (2 b ex vivo) released hydrogen peroxide at a rate of 3.5 1.3 nrnol/min mg protein which was a cyanide-sensitive process. H 2 0 2release from alveolar macrophages decreased slightly but not significantly afer I day in hyperoxia and increased signiJicantly afier 3 days (180%, p < .Or) and 14 days (380%, p < .Ol). When H 2 0 , release was expressed as nmol from total macrophages per animal, the increase afer 14 days in hyperoxia was 760%. H,U, generation by hyperoxic macrophages was cyanide resistant, indicating the involvement of active NADPH oxidase. In both control and hyperoxic macrophages H,O, release could be significantly stimulated with phorbol myristate acetate @%!A). Comparisons of H,O, release by freshly isolated alveolar macrophages and alveolar type 11 cells must be cautiously intwpreted because some cell functions may change during the isolation procedure. Freshly isolated (6 h ex vivo) control alveolar type II cells were found to generate H,O, at a rate of 0.26 f 0.05 nmol/min mg protein-'. In type 11 cells H2U2release, calculated as nmol/mg protein, decreased during the first 7 days of hyperoxia to 10% (p < .OI) of the control value and then returned back up to the control level afer 14 days. A similar decrease was observed ;f H,O, release was calculated as nmol/cell number. H,O, release from control and hyperoxic type II cells was cyanide sensitive. The decrease in H z 0 2release in type 11 cells was associated with cell membrane injury (as assessed by electron microscopy), while biochemical markers of cellular injury firpan blue exclusion and cellular high-energy phosphates ATE ADP) were unchanged. The ability of type I1 cells to scavenge extracellular H,O, did not change in acute hyperoxia, but it increased signlficantly during the second week in hyperoxia. These results indicate that macrophages but not type 11 cells are stimulated to produce H 2 0 2during prolonged exposure to hyperoxia.

*

From the Duke University Medical Center, Durham, North Carolina, and the Department of Pulmonary Medicine, University of Helsinki, Finland. Address correspondence to /. D.Crapo, MD, PO. Box 3177, Duke University Medical Center, Durham, NC 27710, USA.

Experimental Lung Research 18:655-673 (1992) Copyright Q 1992 by Hemisphere Publishing Corporation

655

656

V. L. Kinnula

et al.

Experimental Lung Research 1992.18:655-673.

INTRODUCTION Oxygen radical generation plays an important role in hyperoxia-induced pulmonary injury. Numerous earlier studies have focused on the role of reactive oxygen species generation by phagocytic cells like alveolar macrophages [ 1-41. Recent studies have shown that other lung cells like alveolar epithelial type 11 cells can generate reactive oxygen species 151. Both the mechanisms and the significance of oxygen radical production by alveolar type I1 cells are unclear. The production of reactive oxygen species by alveolar macrophages can be stimulated by numerous cytokines and immunocomplexes [6-81. However, when intact macrophages are briefly exposed to severe hyperoxia or oxidant stress in vitro or in vivo they do not respond by increasing their oxygen radical production. O n the contrary, they lose their respiratory burst activity and phagocytosis, which seems to be associated with a lowered cellular energy state [2, 9-11] and changes in Ca2+influx, membrane peroxidation, and membrane potential [I, 12-15]. Hyperoxia in vivo differs profoundly from hyperoxia or oxidant stress in vitro. It is associated with recruitment of numerous inflammatory cells which may secrete stimulatory cell mediators. Whether macrophages may be stimulated in vivo by the lung inflammation associated with subacute hyperoxia is unclear. It is also unclear how septa1 cells, like alveolar epithelial type I1 cells, respond during hyperoxia in vivo, These cells represent an epithelial lung cell that is relatively resistant against oxidant stress [16] and that can also generate significant amounts of reactive oxygen species [ 5 ] , Most superoxide that is released from activated macrophages is rapidly changed to hydrogen peroxide [4]. Several in vitro studies have also shown that H,O, is one of the most important reactive oxygen species produced by phagocytes and that cytotoxicity of phagocytes is mediated by H,O, in endothelial cells [17], epithelial cells [18], and fibroblasts [19]. Absolute superoxide release and consumption is difficult to assess, whereas comparisons between H,O, consumption and release are possible, To investigate the complex effects of hyperoxia, an in vivo exposure to sublethal hyperoxia for 2 weeks was used so that the lungs would have sufficient time to undergo both destructive and inflammatory phases, yet the injury would be mild enough for the lungs to partially recover from the oxidant stress [16, 201. Hydrogen peroxide generation by alveolar macrophages was investigated in both acute and prolonged hyperoxia. Special emphasis was focused on the oxidant and antioxidant characteristics of alveolar type I1 cells. Extracellular H,O, generation and the ability of cells to scavenge extracellular H,O, were investigated using freshly isolated type I1 cells from control and hyperoxic rats. Experiments with macrophages and type IT cells were conducted using specific activators and inhibitors that are known

H,Oz Release by Lung Cells

657

to have effects on reactive oxygen species generation in alveolar macro-

phages. METHODS Specific pathogen-free, Charles River, CD strain male rats weighing 200250g were exposed to 85% 0, for 1-14 days in polystyrene chambers as described previously [2O]. Control animals were exposed to air at similar flow rates in identical chambers. Animals were provided Purina rat chow ad libitum and kept on a 12-h on/l2-h off light cycle at all times. Chambers were opened for cleaning and feeding the animals every 2 days for 10 min.

Experimental Lung Research 1992.18:655-673.

Preparation of Macrophages and Alveolar Type I1 Cells Solution A for macrophage isolation contained 140 mM NaCl, 5 mM KCl, 8 mM Na,HPO,, 1.4 mM NaH,PO,, 10 mM HEPES (N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid), and 6 mM glucose, pH 7.4, at 22OC. Solution B for type I1 cell isolation contained 140 mM NaCl, 5 mM KCl, 0.5 mM phosphate buffer, 10 mM HEPES, 2.0 mM CaCl,, and 1.3 mM MgSO,. Rats were anesthetized intraperitoneally with an injection of pentobarbital sodium (10 mg/kg body wt) within 1 h after being removed from the chamber. After lung perfusion through the pulmonary artery the lung were lavaged to total lung capacity 8 times with solution A, Each time the lungs were gently massaged and then the lavage fluid was withdrawn slowly. It was filtered through nylon mesh (45 pm) and the pooled lavage fluid was centrifuged at 3008 for 10 min. After one washing the cells were suspended in 1 mL of solution A, Type I1 cells were isolated in solution B by elastase (Worthington Biochemical, NJ) digestion and panning the cell suspensions on IgG pretreated plates for 60 min in Dulbecco’s modified Eagle’s medium at 37°C [21]. The cell preparations were counted using standard hemocytometer techniques. Cell purity and characterization was assessed by light microscopy. Cell viability was measured by assessing the ability of the cells to exclude trypan blue. Because cell culture is known to change the characteristics of type I1 cells within the first 24 h [22, 231, all biochemical assays were conducted immediately after the isolation (1.5-2 h ex vivo for macrophages and 5-6 h ex vivo for type I1 cells). Biochemical Analyses Extracellular H,O, release was measured spectrofluorometrically by follow(hoing the H,O,-dependent oxidation of 3-methoxy-4-hydroxyphenylacetic movanillic acid) to a fluorescent dirner in the presence of horseradish peroxidase using a modification of the method of Ruch et al. [24]. Isolated cells (0.1 x lo6 macrophages, 0.5 x lo6 type I1 cells) were plated on 3.5-cm

Experimental Lung Research 1992.18:655-673.

658

V. L. Kinnula

et

al.

dishes and incubated 15-30 min (macrophages) or 30-60 min (type I1 cells) with homovanillic acid and horseradish peroxidase in HEPES-buffered Hanks' balanced salt solution (HBSS) (pH 7.4) without extracellular CaZt. In these experiments freshly isolated cells in suspension were pipetted on plates and analyzed immediately for H,O, release (within 1.5-2 h ex vivo). Using plated cells, H,O, release was linear during the entire incubation time. It was decreased after 30 min in macrophages. The fluorescence of the supernatant was measured after adjusting the pH to 10 with 0.1 M glycineNaOH buffer (pH 12) (excitation wavelength 321 nm, emission wavelength 425 nm). The exact H,O, concentration of the H,O, solution used to establish a standard curve was determined spectrophotometrically at 240 nm using the extinction value 40 M-'cm-'. Because preliminary experiments showed significant stimulation of hyperoxic macrophages, experiments were also conducted in the presence of Ca + and phorbol myristate acetate (PMA) because macrophages are known to be stimulated with both Caz+and PMA [25]. PMA is known to stimulate macrophages by activation of protein kinase C [4]. We also incubated control and hyperoxic macrophages and type 11 cells with 10 ng/mL PMA without extracellular Ca2+.In additional experiments H,O, release from control and hyperoxic macrophages and type I1 cells was assayed in the presence of 1 mM KCN. Earlier studies have shown that the respiratory burst by macrophages is associated with activation of NADPH oxidase, an enzyme that is cyanide insensitive [26]. Horseradish peroxidase, which was used in the H,O, assay, may bind to KCN [27]. In the present study when 4 U/mL horseradish peroxidase was used, KCN did not influence the detection of H,O,. The H,O, scavenging capacity of type I1 cells was determined by incubating the cells (5 x lo5) in HBSS in the presence of sublethal (30 pM) H,O, [28, 291. Aliquots were drawn for H,O, assays, which were determined as described above. ATP and ADP concentrations from type I1 cells were assayed fluorometrically [30]. Proteins were assayed by the method of Lowry et al. [31], and D N A by the method of Labarca and Paigen [32].

Electron Microscopy Isolated alveolar type I1 cells or macrophages were fixed by suspending the cell pellets in 2% glutaraldehyde in 0.085 M cacodylate buffer (pH 7.4) for 24 h. Cells were pelleted and washed with two changes of phosphatebuffered saline (PBS). Cells were suspended, pelleted in 10% gelatin (37OC), and chilled at 4OC for 30 min to solidify the gelatin. The cell pellets embedded in 10% gelatin were then fixed for 10 min with 2% glutaraldehyde, washed with PBS, and processed for electron microscopic examination by standard procedures [33]. Ultrathin sections were cut with a Reichert ultracut 4E ultramicrotome and picked up on colloidin-coated 200-mesh copper-

659

H,O, Release by Lung Cells

rhodium grids. Sections were stained with uranyl acetate and lead nitrate and examined with a Philips CM 10 electron microscope.

Statistical Analysis Data from various groups were expressed as means f SE. A two-tailed Student t test was used to compare two groups. To compare the significance between multiple experimental groups, two-way analysis of variance (ANOVA) in combination with a post hoc Scheffe test was used. Statistical significance was defined as p < .05.

Experimental Lung Research 1992.18:655-673.

RESULTS The yield of control macrophages was 6.4 f 0.9 x lo6 with >95% purity and viability. Macrophages after 5 and 7 days in hyperoxia were not studied, because the cells in bronchoalveolar lavage were contaminated with a high percentage of inflammatory cells and red blood cells. In this study those macrophage preparations that were > 90% pure were accepted. The yield of macrophages after 1 day in hyperoxia was 4.6 f 0.9 x 106 celldrat, and after 14 days was 22.2 k 2.7 x lo6 (p < .05 compared to controls). The yield of control type I1 cells was 22.6 f 1.4 x lo6 celldrat. After 7 days in hyperoxia the yield was 37.2 f 3.0 x 106/rat (p < .05 compared to controls) and after 14 days the yield was 29.3 f 3.9 x lo6. Type 11 cell yield, purity, and viability data are expressed in Table 1.

Extracellular H,O, Release from Macrophages Unstimulated control macrophages (2 h ex vivo) released H,O, at a rate of 3.5 If: 1.3 nmol H,O,/min mg protein-' in Ca2'-free medium. H,O, release calculated as nmol/min rng protein-' showed a small, nonsignificant tenTable 1 Type I1 Cell Yield, Purity and Viability

Day 0

1 2 5

7 14

n

Yield x 106 Animal Cells

16 3 3 5 6 6

22.6 18.3 20.0 22.8 37.2 29.3

f 1.4 f 2.3 f 4.5 f 0.97 f 3.0'k'' f 3.9

Viability %

Purity

91 f 0.7 92 f 0.1 93 0.3 89 f 3.5 93 f 1.0 95 0.6

89 90 90 84 87 89

*

+_

Note. Cell viability was assessed as trypan blue exclusion. Values are means i SE, n rate isolations. *p < .05 vs 14 days and controls, '"'p < .05 vs controls.

-

OO /

f 0.6 f 1.7 f 0.4 f 0.9'E

f 0.3

f 0.5

number of sepa-

660

V. L. Kinnula et al.

dency to decrease after 1 day in hyperoxia and a significant increase after both 3 days (+ 180%) and 14 days ( + 380%). The change after 14 days was even more prominent ( + 760%) when the results were calculated as H,O, release from total macrophages obtained per animal (Fig. 1). In control macrophages H20,release could be stimulated by adding Ca2+to the incubation medium (going from 3.5 f 1.1 to 5.4 k 1.2 nmollmin mg protein-' (p < ,05) and by PMA (going to 9.7 2.2 nmol/min mg protein-') (p < .05). Macrophages from animals kept in hyperoxia for 14 days showed H,O, release that could be stimulated only with PMA (21.9 f 4.7 to 32.1 f 5.9) (Fig. 2). H,O, release by hyperoxic macrophages was cyanide insensitive, while it was fully inhibited with cyanide in cells from control animals (Fig, 3).

*

Experimental Lung Research 1992.18:655-673.

Extracellular H,O, Release from Type I1 Cells

*

Control type I1 cells (6 h ex vivo) released H,02 at a rate of 0.26 0.05 nmol/min mg protein-'. H,O, release by type I1 cells, calculated as nmol/min mg protein-', decreased significantly during the first week in hyperoxia to 10% of the control values and returned to the control level (p < .1) during the second week (Fig. 4, Ca2+free). Similarly, extracellular H,O, release calculated as nrnol/min cell number-' was decreased in subacute hyperoxia (data not shown). In control cells H,O, release was lowest in Ca2+-freemedium and was increased significantly by PMA (Fig. 4). In hyperoxic cells H,O, release was dramatically decreased in Caz+-freemedium and in the presence of PMA after 7 days but not after 14 days. Control type I1 cells produced H,O, in the presence of PMA at a rate of 0.46 nmol/min mg protein-'; the corresponding values after 7 and 14 days were 0.11 and 0.45 (Fig. 4). Extracellular H,02 release from control and hyperoxic type I1 cells was cyanide sensitive (Fig. 5).

ctl CL,

40

nrnollminltotal macrophages

. . I5

mg protein

0

5

I0

Days in 85% 0 2 Figure 1 H,O, release from isolated macrophages (2 h ex vivo) after 1-14 days exposure to hyperoxia in vivo. 0, H202release when calculated as nmol/min mg protein-'. e, HZO, release when Calculated as nrnol/min coca1 number of macrophages obtained-' animal-'. Values are means f SE from 3-6 separate isolations; " p < .05 compared to controls.

Experimental Lung Research 1992.18:655-673.

H,O, Release by Lung Cells

661

Figure 2 H20, release from control and h eroxic (14 days) macrophages. Measurements were made on isolated macrophages (2 h ex vivo) in C>'-free HBSS, in the presence of 1.3 m M Ca2+ and in the presence of 10 ng/mL PMA. H 2 0 2 release is expressed as nmol/min mg protein-'. Values are means f SE from 3-5 separate isolations; '+p < .05 compared to the PMA group, **p < .05 compared to both the Ca" and the PMA groups. The differences berween control and hyperoxic groups were significant in each case.

Because lungs are damaged in subacute hyperoxia after 7 days [16] and because freshly isolated type I1 cells are unstable as to their H202generation [5], we evaluated the magnitude of cell injury during hyperoxia and/or during the cell isolation using tests of cell viability and cellular energy states. Type I1 cell viability was the same after 7 and 14 days and did not differ from the control level (trypan blue exclusion) (Table 1). Because changes in cell viability occur later than do changes in cellular energy states [lo], intracellular ATP and ADP concentrations in type I1 cells after 7 days in hyperoxia were measured. These assays, which were conducted in duplicates from two separate cell isolations, showed that ATP and ADP concentrations in control cells were 0.022 and 0.039 pmol/mg DNA. The corresponding values after 7 days in hyperoxia were 0.022 and 0,040 pmol/rng, respectively.

Electron Microscopy Type I1 cell characteristics were also investigated using electron microscopy to look for ultrastructural indications of cell injury and, in particular, cell membrane integrity. Ultrastructural characteristics of the cell populations were similar in control and hyperoxic type I1 cells after 7 days. No overt indications of cell injury were observed. Type I1 cells from 14-day hyperoxic animals contained a large number of mitochondria, Both control and hyperoxic type I1 cells had lamellar bodies densely packed with myelin (Figs. 6-8). No obvious change in ultrastructure of mitochondria1 membranes or endoplasmic reticulum in hyperoxic type I1 cells could be observed, which is also in agreement with the finding that the concentration of high-energy phosphates was unchanged in hyperoxic cells. However, in hyperoxic type

662

V. L. Kinnula et al.

A

with 1mM KCN

c

1

0

10

.

20

30 Time, min

B

Experimental Lung Research 1992.18:655-673.

500

-c

400

7

-

Hyproxic macrophages

without KCN

0)

c.

g 300-

=E 0

200-

5

0

10

20

30

Tlme, mln Figure 3 The effects of 1 mM KCN on H20, release from freshly isolated macrophages in Ca2+-free HBSS isolated from ( A ) control animals and (B) animals exposed to hyperoxia for 14 days. Values are means f SE from 3 separate isolations; 'kp < .05 compared to cells with 1 mM KCN.

I1 cells after 7 days, but not after 14 days, plasma membranes were focally swollen and disrupted. These changes were observed in most cells; a typical change is shown in Fig. 9. This phenomenon was associated with leakage of protein-like material to the extracellular space. In addition, the plasma membrane profile of type I1 cells after 7 days exposure was less sharp than it was in control cells and hyperoxic cells after 14 days exposure. This gives the impression of plasma membrane changes throughout the cell surface of type I1 cells exposed to hyperoxia for 7 days. We also compared the ultrastructural characteristics of macrophages in cell pellets recovered from lavage of air- and 85% 0,-(14 days) exposed rats. The macrophages from air-exposed rats were a homogenous population of cells with a dense cytoplasmic matrix and modest numbers of uniformly

663

Experimental Lung Research 1992.18:655-673.

H202Release by Lung Cells

Figure4 Extracellular H20, release from control and hyperoxic type I1 cells after 7 and 14 days. Measurements were made in HBSS without CaZ', with 1.3 mM Ca2+, and with 10 ng/mL PMA. H202 release is expressed as nmol/min mg protein-'. Values are means from 3-5 separate isolarions; "p < .05 compared to PMA, ""p < .05 compared to both the CaZf and the PMA groups. The values for the cells exposed in vivo to hyperoxia for 7 days were significantly lower than the corresponding values for both control cells and cells exposed to hyperoxia in vivo for 14 days, while the latter two sets were not different from each orher.

20

Control type II cells

15

,o{

0

10

20 30 T h e , mln

Hyperoxic

"1

/.

p i t h o u t KCN

type II cells

p

/'without without KCN

oy-:

with 1rnM KCN with 1rnM KCN

0

10

20 30 Time, min

Figure 5 The effects of 1 mM KCN on HzOzrelease from freshly isolated type I1 cells from (A) control animals and (B) animals exposed to hyperoxia in vivo for 14 days. Values are means from 2 separate isolations.

Experimental Lung Research 1992.18:655-673.

664

V. L. Kinnula et al.

-

Figure 6 Alveolar type I1 cell isolated from control rat lung. Nu, nucleus; m, mitochondria; Ib, lamellar bodies. Bar 2 urn.

distributed mitochondria and phagolysosomes. Macrophages isolated from oxygen-exposed rats were significantly larger. The majority of these cells displayed a more electron lucent cytoplasmic matrix than did macrophages isolated from control animals. The numbers and sizes of phagolysosomes in these macrophages were increased, although they varied greatly between cells. In addition, greater than 50% of the macrophages from 0,-exposed rats showed a greatly induced density of mitochondria (Fig. 10).

665

H,Oz Release by Lung Cells

H,O, Scavenging Capacity by Isolated Type I1 Cells

Experimental Lung Research 1992.18:655-673.

The capacity of type I1 cells to scavenge extracellular H,O, was measured by incubating the cells with 30 pM H,O, for 5-20 min. In cells from hyperoxicconsumption could be exposed animals no change in the extracellular HzOz observed after 1 day, while after 7 days H,O, consumption from the extracellular medium was increased minimally (data not shown) and after 14 days it was increased significantly (Fig. 11).

Figure 7 Alveolar type I1 cell isolated from rat lungs exposed to 85% oxygen for 7 days. Bar

-

2 pm.

Experimental Lung Research 1992.18:655-673.

666

V. L. Kinnula et al.

Figure 8 Alveolar type I1 cell isolated from rat lungs exposed to 85% for 14 days. The ultrastructure of

-

these cells is similar to that of cells from air-exposed rats except chat the cells contained a higher density of subcellular organelles such as mitochondria. Bar 2 I m .

DISCUSSION

The present study showed that hydrogen peroxide generation by alveolar macrophages is stimulated by exposure to prolonged hyperoxia. Earlier results have shown that the number of inflammatory cells in the lung increases after 5 days in 85% 0, [16, 341. Inflammatory cells are known to produce high amounts of oxygen radicals and secrete cell mediators that activate or stimulate the cells further. After 7 days in 85% oxygen the volume of neutrophils in the lung interstitiurn returns toward normal [34]. In the present study extracellular H,O, release from macrophages increased within 3 days and was even higher after 2 weeks when calculated as nmol

H20, Release by Lung Cells

667

H,02 released from the total macrophages obtained. Earlier studies have

Experimental Lung Research 1992.18:655-673.

shown that alveolar macrophages are heterogenous and contain many subpopulations [35, 361. It is likely that the macrophage population changes during a hyperoxic exposure, which is supported by the structural changes observed in these cells using electron microscopy. Either way the presence of increased numbers of activated macrophages is likely to contribute to continued oxidant stress against other lung cells after 2 weeks in hyperoxia. Macrophages are known to be stimulated to produce reactive oxygen

Figure 9 Representative type I1 cell plasma membrane after 7 days in hyperoxia. In a large number of type I1 cells the plasma membrane was obviously damaged. These areas were associated with increased amounts of protein-like material (*) in the adjacent extracellular space suggesting leakage through damaged plasma membranes. In addition, the plasma membrane had lost its sharp detail (arrow heads). The loss of plasma membrane detail was not found in control cells or in cells exposed t o hyperoxia for 14 days. M, mitochondria; Ib, lamellar bodies; rer, rough endoplasmic reticulum, r, ribosomes. Bar 0.5 pm.

..

Experimental Lung Research 1992.18:655-673.

668

V. L. Kinnula et al.

Figure 10 Ultrastructural characteristics of isolated rat alveolar macrophages: (A) control animal; (B) after exposure in vivo t o 85% 0, for 7 days. Note the increased cell size after 0 2 ex osure and the increase in the numbers of mitochondria and phagolysosomes. N, nucleus; M, mitochonJia; P,phagolysosome. Bar 2.0 pm.

-

species by inflammatory mediators like interleukins, interferons, and tumor necrosis factor [3, 6, 71, but they are not stimulated by acute hyperoxia in vitro or in vivo [l, 9, 121. Furthermore, reactive oxygen species generation by these cells is known to be saturated by a low oxygen tension [37]. In the present study macrophages were not stimulated by acute hyperoxia, but they were stimulated by hyperoxia after 3 days. This is also the time re-

Hyperoxlc Control

UllO

400

0

5 101520 Incubation t h e , mln

Figure 11 Ability of type I1 cells to scavenge extracellularly added H,O,. The cells were freshly isolated (6 h ex vivo) from either control animals or animals exposed to hyperoxia in vivo for 14 days. Cells were incubated in the presence of 30 pM H,O,. AIiquots for H,O,, assays were drawn during a 20-min incubation period. Values are means f SE from 3 separate isolations; “p < .05 when compared t o the corresponding control value.

Experimental Lung Research 1992.18:655-673.

H202Release by Lung Cells

669

quired for the first inflammatory changes in lungs during a normobaric hyperoxic exposure [34]. The time course of macrophage stimulation suggests that these cells may not be stimulated by hyperoxia per se. Stimulation of alveolar macrophages is associated with the inflammatory phase and probably involves recruitment of new populations of macrophages into the alveolar spaces. Stimulation of reactive oxygen species generation by alveolar macrophages is associated with mobilization of intracellular calcium [4]and by activation by cyanide resistant NADPH oxidase [26] through the protein kinase C pathway. Reactive oxygen species generation by alveolar macrophages is known to be stimulated with PMA which activates protein kinase C in these cells [4].In the present study H,O, release by hyperoxic alveolar macrophages was also cyanide resistant, which indicates that after exposure to hyperoxia NADPH oxidase was activated in these cells. H,O, generation by hyperoxic macrophages could not be stimulated with CaZ+,in contrast to control cells where Caz+gave a mild degree of stimulation. Hyperoxic macrophages could still be stimulated with PMA. This may be due to an increase in protein kinase C activity or to a changed macrophage population in response to hyperoxia. H,O, release from macrophages and type I1 cells is difficult to compare. We have recently shown that freshly isolated macrophages and type I1 cells are unstable, with H20, release from the freshly isolated cells decreasing rapidly ex vivo [S]. When H202release assays have been conducted at the same time point (5-6 h ex vivo) unstimulated macrophages produce H,O, at a rate that is 4 to 5 times higher than that from type I1 cells [5]. Because freshly isolated cells are unstable and type I1 cells change rapidly in culture [2O],in this study all experiments were conducted immediately after the cell isolation (2 h ex vivo for macrophages and 5-6 h for type I1 cells). Although absolute comparisons between these isolated cells are difficult, the results of both previous studies [5] and the present study clearly show that unstimulated macrophages release significantly more H,O, than do type I1 cells. Both freshly isolated and cultured alveolar epithelial type I1 cells have been observed to generate reactive oxygen species extracellularly [5, 381, although the mechanism of reactive oxygen species generation is unclear. Furthermore, earlier studies with aminotriazole and BCNU, which inactivate catalase and glutathione reductase, suggest that H,O, is released from the cell membrane andlor is not accessible to intracellular antioxidant enzymes j5] in these cells. This study showed that extracellular H202release could be stimulated with PMA and that it decreased dramatically between 3 and 7 days in hyperoxia. This decreased H,O, release was observed at the same time when the antioxidant capacity, assessed as a rate of scavenging extracellular H202,was slightly increased. However, after 14 days, H,O, release was the same as in controls and H,O, scavenging capacity was higher than it was after 7 days. These results indicate that the net extracellular

Experimental Lung Research 1992.18:655-673.

670

V. L. Kinnula et al.

H202release rate does not directly correlate with the antioxidant characteristics in alveolar type I1 cells, The results of the present study may also suggest possible involvement of protein kinase C in the regulation of H202 release from alveolar epithelial type 11 cells. Increased H20,consumption by type I1 cells in hyperoxia may reflect cell proliferation and/or increased antioxidant enzyme activities in these cells. Earlier studies have shown that type I1 cells proliferate within the first week in hyperoxia [39]. Furthermore superoxide dismutase and catalase specific activities increase after the first week [39]. Others have shown that type 11 cells scavenge extracellular H,02 mainly by a catalase-dependent pathway [38, 401. In the present study H,02 consumption did not change during the first week but increased significantly after 14 days. This time course is consistent with earlier published data about increased antioxidant enzyme specific activities in hyperoxia [39] and indicates an increased antioxidant capacity in hyperoxic type I1 cells. An initial decrease in reactive oxygen species generation was found to occur in freshly isolated type I1 cells and may be related to cell injury occurring during hyperoxia and/or cell isolation. Morphologic studies have shown that lungs are damaged in sublethal hyperoxia (85% 0,)after 5 and 7 days, [16,411. Hyperoxia causes dramatic changes in the subcellular composition of both type I and type I1 pneumocytes. The volume densities of almost every type of cell substructure increase in epithelial pneumocytes during hyperoxia. Furthermore, a ruffled air border in type I cells, indicating membrane injury, can be observed [41]. To analyze if the decrease in H,O, generation by hyperoxic type I1 cells in the present study is associated with cell injury, conventional biochemical methods and electron microscopy were used. Membrane discontinuities, which were observed in type 11 cells, were focal and biochemical markers of cellular injury (trypan blue exclusion and intracellular high-energy phosphates) remained unchanged. These results suggest that membrane damage may affect the rate of H,O, release from type I1 cells. Despite numerous subcellular changes in hyperoxic (85% 0,)type I1 cells in situ [41], no observations of type I1 cell plasma membrane injury in situ have been reported. Extrapolation of the results of in vitro studies to the situation in vivo must be done with caution. Furthermore, during the isolation procedure aggravation of existing subtle membrane injury may occur. One can argue that the results obtained with type I1 cells are due to macrophage-contaminated cell preparations. Macrophages are known contaminants of type I1 cell preparations and they produce more reactive oxygen species than do type I1 cells. It has to be emphasized, however, that when H,O, assays are conducted at the same time ex vivo (6 h) for both cell types, unstimulated macrophages produce 4 to 5 times more H,O, than do type I1 cells [5]. In the panning technique used in this study most macrophages can be attached to IgG pretreated plates and separated from type I1

Experimental Lung Research 1992.18:655-673.

H,02 Release by Lung Cells

671

cells [21]. Complete separation of macrophages from type I1 cells is not possible using current protocols. Type I1 cell preparations contain approximately 3-4% macrophages [5, 22). However, even this amount of macrophages, if stimulated, could result in an increased H,O, release by type I1 cell preparations. This is especially true in hyperoxic type I1 cells after 2 weeks because macrophages were increased in number and were stimulated to produce more H,O,. In the present study H,O, generation by hyperoxic type I1 cells was cyanide sensitive and it was not stimulated by PMA. This is opposite to what would have been expected if our cell preparations had significant macrophage contamination. The results of the present study indicate that H,O, generation by type I1 cells was not significantly influenced by macrophages. In summary, the present study showed a significant increase in H,O, release by alveolar macrophages in sublethal hyperoxia which was cyanide insensitive, indicating an active NADPH oxidase in these cells. Also, type I1 cells released low amounts of H,O, t o the extracellular space, This release of H,O, by type I1 cells could be stimulated by PMA and was cyanide sensitive. H,O, release from type I1 cells was impaired in subacute hyperoxia, possibly related to the type of cell injury. The results also suggest that increased reactive oxygen species generation from stimulated inflammatory cells like alveolar macrophages can partly explain lung cell damage during inflammation and oxidant stress, This work was supported by NIH Program Project Grant PO1 HL31992 and by R01 HL-42609.

WFERENCES 1. Forman HJ, Nelson J, Harrison G: Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production. J Appl Physiol 60:1300-1305, 1986. 2. Forman HJ, Dorio RJ, Skelton DC: Hydroperoxide-induced damage to alveolar macrophage function and membrane integrity: alterations in intracellular-free Car+ and membrane potential. Arch Biochem Biophys 259:457-465, 1987. 3. Sibille Y, Reynolds Y: Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Respir Dis Rev 141:471-501, 1990. 4. Rossi J: The 0;-forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 853:65-83, 1986. 5. Kinnula VL, Everitt JI, Whorton AR, Crapo JD: Hydrogen peroxide production by freshly isolated alveolar type 11 cells, alveolar macrophages and cultured endothelial cells. Am J Physiol: Lung Cell & Mol Physiol 261:L84-L91, 1991. 6. Nathan CF, Murray HW, Wiebe ME, Rubin BY: Identification of interferon-h as the lymphokine metabolism and antimicrobial activity. J Exp Med 158670689, 1983. 7. Fels AOS, Nathan CF, Cohn ZA: Hydrogen peroxide release by alveolar macrophages from sarcoid patients and by alveolar macrophages from normals after

Experimental Lung Research 1992.18:655-673.

6 72

V. L. Kinnula et al.

exposure to recombinant interferons alpha, beta and gamma and 1,25-dihydroxy vitamin D,. J Clin Invest 80:381-387, 1987. 8. Ward PA, Duque RE, Sulavik MC, Johnson KJ: In vitro and in vivo stimulation of rat neutrophils and alveolar macrophages by immune complexes. Am J Pathol 110297-309, 1983, 9. Suttorp N, Simon LM: Decreased bactericidal function and impaired respiratory burst in lung macrophages after sustained in vitro hyperoxia. Am Rev Respir Dis 128:486-490, 1983. 10. Sutherland MW, Nelson J, Harrison G, Forman HJ: Effects of t-butyl hydroperoxide on NADPH, glutathione and the respiratory burst of rat alveolar macrophages. Arch Biochem Biophys 243:325-331, 1985. 11. Oosting RS, Van Bree L, Van Iwaarden JF, Van Golde LMG, Verhoef J: Impairment of phagocytic functions of alveolar macrophages by hydrogen peroxide. Am J Physiol 259 (Lung Cell Mol Physiol 3):L87-L94, 1990. 12. Wolff LJ, Boxer LA, Allen JM, Baehner RL: The selective effect of hyperoxia on the guinea pig alveolar macrophage membrane. J Reticuloendoth SOC 24:377381, 1978. 13. Simon LM, Axline SG, Robin ED: The effect of hyperoxia on phagocytosis and pinocytosis in isolated pulmonary macrophages. Lab Invest 39:541-546, 1978. 14. Forman HJ, Williams JJ, Nelson J, Daniele RP,Fisher AB: Hyperoxia inhibits stimulated superoxide release by rat alveolar macrophages. J Appl Physiol (Respir Environ Exercise Physiol) 53:685-689, 1982. 15. Forman HJ, Skelton DC: Relationship of depolarization to inhibition of the alveolar macrophage respiratory burst by H,O,. Free Radic Biol Med 9:141, 1990. 16. Crapo JD, Barry BE, Foscue HA, Shelburne J: Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am Rev Respir Dis 122:123-143, 1980. 17. Weiss SJ, Young J, LoBuglio F, Slivka A, Nimeh NR: Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J Clin Invest 68:714-72 1, 1981. 18. Suttorp N, Simon LM: Lung cell oxidant injury: enhancement of polymorphonuclear leucocyte-mediated cytotoxicity in lung cells exposed to sustained in vitro hyperoxia. J Clin Invest 70:342-350, 1982. 19. Simon RH, Scoggin CH, Patterson D: Hydrogen peroxide causes fatal injury to human fibroblasts exposed to oxygen radicals. J Biol Chem 256:7181-7186, 1981. 20. Crapo JD: Morphological changes in pulmonary oxygen toxicity. Annu Rev Physiol 48:721-731, 1986. 21. Dobbs LG, Gonzalez R, Williams MC: An improved method for isolating type cells in high yield and purity. Am Rev Respir Dis 134:141-145, 1985. 22, Dobbs LG: Isolation and culture of alveolar type I1 cells. Am J Physiol 258 (Lung Cell Mol Physiol 2) L134-L148, 1990. 23. Finkelstein JN: Physiologic and toxicologic responses of alveolar type cells. Toxicology 60:41-52, 1990. 24. Ruch W, Cooper PH, Baggrolini M: Assay of Hz02production of macrophages

H20, Release by Lung Cells

25.

26. 27.

28.

29.

Experimental Lung Research 1992.18:655-673.

30. 31. 32. 33. 34.

35.

36. 37. 38.

39.

40.

41.

6 73

and neutrophils with homovanillic acid and horseradish peroxidase. J Immunol Methods 63:347-357, 1983. Dorio RJ, Nelson J, Forman HJ: A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages. Biochim Biophys Acta 928:137-143, 1987, Bellavite P, Serra MC, Davoli A, Bannister JV, Rossi F: The NADPH oxidase of guinea pig polymorphonuclear leucocytes. Mol Cell Biochem 52:17-25, 1983. Cotton ML, Dunford HB, Raycheba JMT: Studies of horseradish peroxidase, VIII: the kinematic effect of cyanide on the oxidation-reduction cycle. Can J Biochem 51:627-631, 1973. Whorton AR, Montgomery ME, Kent RS: Effect of hydrogen peroxide on prostaglandin production and cellular integrity in cultured porcine aortic endothelial cells. J Clin Invest 76:295-302, 1985. Spragg RG: DNA strand break formation following exposure to bovine pulmonary artery and aortic endothelial cells to reactive oxygen products. Am J Respir Cell Mol Biol 4:4-10, 1991. Lowry OH, Passonneau JV: A Flexible System of Enzymatic Analysis. New York: Academic, pp 152-156, 1972. Lowry OH, Rosebrough NR, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951. Labarca C, Paigen K: A simple, rapid and sensitive DNA assay procedure. Anal Biochem 102:344-352, 1980. Glauert AM: Fixation and Embedding of Biological Specimen. New York: American Elsevier, 1975. Barry BE, Crapo JD: Patterns of accumulation of platelets and neutrophils in rat lungs during exposure to 100% and 85% oxygen. Am Rev Respir Dis 132:548-555, 1984. Shellito J, Kaltreider HB: Heterogeneity of immunological function among subfractions of normal rat alveolar macrophages. Am Rev Respir Dis 129:747-753, 1984. Brannen AL, Chandler DB: Alveolar macrophages subpopulations’ responsiveness to chemotactic stimuli. Am J Pathol 132:161-166, 1988. Gabig TG, Bearman SI, Babior BM: Effort of oxygen tension and p H on the respiratory burst of human neutrophils. Blood 53: 1133-1 139, 1979. Kinnula VL, Chang LY, Everitt JI, Crapo JD: Oxidants and antioxidants in alveolar epithelial type I1 cells: in situ, freshly isolated and cultured cells. Am J Physiol (Lung Cell Mol Physiol), 262:169-177, 1992. Freeman BA, Mason RJ, Williams MC, Crapo JD: Antioxidant enzyme activity in alveolar type I1 cells after exposure of rats to hyperoxia. Exp Lung Res 10~203-222, 1986. Engstrom PC, Easterling CL, Baber RR, Matalon S : Mechanisms of e x t r a c e h lar hydrogen peroxide clearance by alveolar type I1 pneumocytes. J Appl Physiol 69:2078-2084, 1990. Harris JB, Chang LY, Crapo JD: Rat lung alveolar type I1 epithelial cell injury and response to hyperoxia. Am J Respir Cell Mol Biol 4:115-125, 1991.